The present invention relates to novel disaccharides, the solvates and pharmaceutically acceptable salts thereof, and to the pharmaceutical compositions containing them. This invention also refers to a process for preparing the novel disaccharides, as well as to the therapeutical use thereof.
Arthrosis (osteoarthritis) is the most common articular rheumatic disease, affecting most people over 65 years of age, characterised by a gradual degradation of the cartilaginous tissue, together with the presence of inflammation and pain. The term arthrosis describes a disease in which the hyaline cartilage of the articulations is destroyed.
At present, therapy is centred on relieving the symptoms, since no agent has yet been found that provides a proven reduction of the progression of the damage to the cartilage, although there is some clinical evidence for chondroitin sulphate and hyaluronic acid.
The substances that act on the symptoms include: rapid action substances such as analgesics, non steroidal anti-inflammatory drugs (NSAIDS) and corticoids, and substances that have a somewhat slower effect, known as SYSADOA (Symptomatic slow acting drug for osteoarthritis) (M. G. Lequesne, Rev. Rhum. (Eng./Ed.), 61, 69-73 (1994)), which include hyaluronic acid, chondroitin sulphate and glucosamine sulphate.
Symptomatic slow acting drugs have the additional advantage that they are safer than NSAIDS (E. Maheu, European Journal of Rheumatology and Inflammation, 15, 17-24 (1995)), and have longer-lasting effects, which even persist for some months after the cessation of treatment.
Recently, clinical trials conducted with hyaluronic acid (V. Listrat et al., Osteoarthritis Cart., 5, 153-160 (1997)) and with chondroitin sulphate (G. Verbruggen et al., Osteoarthritis Cart., 6 (Supplement A), 37-38 (1998)) have for the first time provided evidence of the possibility that these two compounds, besides acting as SYSADOA, may influence and delay the course of the arthrosic disease, (chondro-protective agents).
Hyaluronic acid is a non-sulphate glycosaminoglycan of natural origin, with a polymeric structure composed of disaccharides of N-acetylglucosamine and glucuronic acid.
Hyaluronic acid is extracted from mammal organs and/or tissues. One known problem lies in the fact that, depending on how it is obtained, the molecular weight of the product can vary, which, together with the fact that it may come from different sources, means that there are several hyaluronic acids that may or may not have the same clinical effects.
The disaccharides of the present invention are structurally related to the dimers present in the polymeric structure of hyaluronic acid, in as much as they are disaccharides with xcex2-(1xe2x86x923) unions between the glucuronic acid and the glucosamine, but the disaccharides of the present invention always contain a sulphate group in the C-4 and/or the C-6 of the glucosamine ring.
Some compounds have been described in the bibliography that can also be considered to be structurally related to the compounds of the present invention.
J. R. Couchman et al. (EP 211610) disclose esterified disaccharides that differ from the compounds of the present invention in the nature of the alkyl root of the ester group (xe2x80x94COORxe2x80x2). These compounds also differ from those of the present invention in that they are useful for stimulating hair growth and for the treatment of baldness.
The disaccharides that are repeated in the structure of chondroitin sulphate, the sulphated derivative both in position 4 and position 6 of the N-acetylgalactosamine, are commercially available, and they are obtained by degradation of the natural polymers or by chemical synthesis (J. C. Jacquinet, Carbohydrate Research, 199, 153-181 (1990); J. C. Jacquinet et al. Carbohydrate Research, 314, 283-288 (1998)), but they differ from the compounds of the present invention in that they contain galactosamine instead of glucosamine. The biological activities of these disaccharides have not been described to date.
Hartung et al. (WO 9309766) disclose a method for treating human and horse painful arthopathic conditions, administering parenterally, intramuscularly, or transdermally an effective amount of a composition containing at least one chondroitin sulfate salt. In the same manner, Nocelli et al. (EP 704216) disclose a gel-like pharmaceutical composition containing chondroitin sulfate salts for the treatment of arthrosis by means of an oral administration. The chondroitin derivatives described by Hartung et al. and Nocelli et al. differ from the compounds of the present invention in that they refer to a polymeric structure and that they contain a galactosamine moiety instead of a glucosamine.
Therefore, it is evident that the obtention of new compounds for the treatment of arthrosis and its symptoms, such as inflammation and pain, is still a problem in therapy.
The present invention provides new disaccharides of formula (I), 
in which:
R1 is-selected from the group consisting of: hydrogen, linear or branched (C1-C4)-alkyl, phenylalkyl of less than ten carbon atoms and xe2x80x94COCH3;
R2 is selected from the group consisting of: hydrogen, xe2x80x94COCH3 and SO3M;
R3 is selected from the group consisting of: hydrogen, linear or branched (C1-C4)-alkyl, phenylalkyl of less than ten carbon atoms, xe2x80x94COCH3 and xe2x80x94COPh, where Ph is phenyl;
G is selected from between xe2x80x94COOR4 and xe2x80x94COOM, where R4 is selected from the group consisting of: hydrogen, (C1-C2)-alkyl and arylalkyl of less than sixteen carbon atoms;
A is selected from the group consisting of: hydrogen, xe2x80x94SO3H, xe2x80x94SO3M and xe2x80x94COCH3; and
B is selected from the group consisting of: hydrogen, xe2x80x94SO3H, xe2x80x94SO3M, and xe2x80x94COCH3, where either A or B is necessarily either xe2x80x94SO3H, or xe2x80x94SO3M, and where M is an organic or metallic cation.
The invention also includes the solvates and the pharmaceutically acceptable salts of the compounds of formula (I):
The compounds of formula (I) have an anomeric carbon in their structure. This invention includes anomeric forms xcex1 andxcex2, and their mixtures.
In a preferred embodiment, the compounds of formula (i) are those where: G is xe2x80x94COOR4, or xe2x80x94COOM, where R4 is (C1-C2)-alkyl, or arylalkyl of less than sixteen carbon atoms, and M is a metallic cation.
More preferred are the compounds of formula (I) where: R1 is hydrogen, R2 is xe2x80x94COCH3 and R3 is hydrogen. Equally preferred are the compounds of formula (I) where: R1 is methyl, R2 is xe2x80x94COCH3 and R3 is hydrogen.
Even more preferred are the compounds of formula (I) where A is hydrogen and B is xe2x80x94SO3M, or where A is xe2x80x94SO3M and B is hydrogen, or where A and B are xe2x80x94SO3M, and M is a metallic cation.
Especially preferred are the compounds of formula (I) where: M is the sodium cation.
Particularly especially preferred embodiments of this invention are those in which the compounds of formula (I) are one of the following:
methyl 2-acetamido-2-deoxy-3-O-(xcex2-D-glucopyranosyluronic acid)-6-O-sulfo-xcex1-D-glucopyranoside, disodium salt;
methyl 2-acetamido-2-deoxy-3-O-(xcex2-D-glucopyranosyluronic acid)4-O-sulfo-xcex1-D-glucopyranoside, disodium salt;
methyl 2-acetamido-2-deoxy-3-O-(xcex2-D-glucopyranosyluronic acid)4,0-di-O-sulfo-xcex1-D-glucopyranoside, trisodium salt;
2-acetamido-2-deoxy-3-O-(xcex2-D-glucopyranosyluronic acid)-6-O-sulfo-D-glucopyranose, disodium salt;
2-acetamido-2-deoxy-3-O-(xcex2-D-glucopyranosyluronic acid)4-O-sulfo-D-glucopyranose, disodium salt;
2-acetamido-2-deoxy-3-O-(xcex2-D-glucopyranosyluronic acid)4,6-di-O-sulfo-D-glucopyranose, trisodium salt.
Another embodiment of this invention is a process for preparing a compound of formula (I).
According to this invention, compounds of general formula (I) are obtained by a process characterised in that a monosaccharide of formula (II), 
where R5 represents a reactive group that can establish a xcex2-(1xe2x86x923) union with the free hydroxyl in the monosaccharide of formula (III), R6 can be equivalent to group R4 in (I) or a group that protects carboxyl groups that can be eliminated later, P1, p2 and p3 represent groups that protect hydroxyls that can be eliminated later or they can be equivalent to R3 in (I), is made to react with a monosaccharide of formula (III), 
where R7 can be equivalent to R1 in (I) or it can be a group that may coincide or not with R1, and later be eliminated so that R1xe2x95x90H in (I), R8 can be a group equivalent to R2 in (I) or a group that protects amino groups, P4 and P5 can be protective groups, jointly constituting a cyclic protective group, either P4 or P5 can be acetyl, in which case P4 will be equivalent to B or P5 will be equivalent to A, to form a intermediate disaccharide of formula (IV): 
When reactive group R5 in (II) is Br, then the reaction between (II) and (III) can take place, for example, in a suitable organic solvent, preferably di-dichloromethane, at a suitable temperature, preferably room temperature, and in the presence of a catalyst, such as, for example, silver triflate, and a proton acceptor can also be present.
When reactive group R5 in (II) is xe2x80x94Oxe2x80x94C(xe2x95x90NH)xe2x80x94CCl3, the condensation reaction between the xcex1-imidate (II) and the alcohol (III) can take place in the presence of trimethylsilyl triflate, in a suitable organic solvent, for example dichloromethane, and preferably at room temperature.
Preferably, the p1, p2 and P3 groups represent pivaloyl, benzoyl, acetyl or benzyl groups.
Groups p4 and P5 preferably represent a cyclic protective group like benzylidene, which comes from the reaction of hydroxyls 4 and 6 of the glucosamine ring with the benzaldehyde.
Group R6 preferably represents methyl.
Group R7 preferably represents methyl or benzyl.
Group R8 preferably represents acetyl, trichloroacetyl or benzyloxycarbonyl (BOC).
The intermediate disaccharide of formula (IV) is selectively disprotected. For example, if hydroxyls 4 and 6 of the glucosamine ring are protected in the form of acetal, the disaccharide of formula (IV) can be treated for example with ethanethiol/dichloromethane in the presence of p-toluenesulphonic acid, obtaining the intermediate disaccharide of formula (V), 
where p1, p2, p3, R6, R7 and R8 represent the previously described groups.
From the intermediate disaccharide of formula (V), the functional groups desired can be progressively introduced.
For example, for O-sulphonation in the C-4 of the glucosamine ring, the primary alcohol in position 6 can be protected for example with an acetyl group (in which case CH2OP5 will be obtained, where P5xe2x95x90acetyl), by adding acetic anhydride in a suitable solvent such as pyridine, and a temperature of below 0xc2x0 C., or with a benzoyl group, using for example benzoyl cyanide in, for example, pyridine, and then the free hydroxyl in the C-4 can be made to react with, for example, the sulfur trioxide-trimethylamine complex (SO3-NMe3) in, for example, N,N-dimethylformamide and at a suitable temperature, preferably between 40 and 60xc2x0 C.
For O-sulphonation of the hydroxyl in C-6 of the intermediate disaccharide of formula (V) to take place, it is treated with, for example, the sulfur trioxide-trimethylamine complex (SO3-NMe3) in, for example, N,N-dimethylformamide, obtaining a majority of the sulphate derivative in C-6, together with a smaller proportion of the disulphate derivative. Adjusting the reaction conditions it is possible to increase the proportion of the disulphate derivative (in C-4 and in C-6 of the glucosamine ring).
Once the sulphate groups have been introduced into C-4 and/or C-6 of the glucosamine ring, if groups p1, p2, p3, p4, p5, R6, R7 and R8 are not equivalent to the relevant groups in formula (I), total or selective disprotection will be applied to obtain the compounds of formula (I), and when required, after the disprotection, the group or groups will then be made to react to obtain the compounds of formula (I).
For example, to obtain a formula (I) compound in which R2 is SO3M, once the sulphate group has been introduced into C-4, or C-6, or C-4 and C-6, if R8 is, for example, benzyloxycarbonyl (BOC), hydrogenolysis will be performed, and the amino group obtained will later be made to react at basic pH with, for example, the sulfur trioxide-trimethylamine complex.
To obtain a compound of formula (I) in which R1 is hydrogen, it is possible to start with another compound of formula (I) in which R1 is benzyl, and then remove the benzyl group by hydrogenolysis, using, for example, Pdxe2x80x94C in the presence of a suitable solvent such as, for example, aqueous methanol.
To illustrate the described procedure, Schemes 1 and 2 show the synthetic sequences to obtain some compounds of formula (I) 
Moreover, to also illustrate the described procedure, Scheme 3 shows the synthetic sequence to -obtain some compounds of formula (I) in which R1 is hydrogen or benzyl. 
The initial monosaccharide of formula (II) can be obtained from the commercial compound D-glucurono-3,6-lactone of formula (IX), by means of a sequence of reactions that are well known in carbohydrate chemistry.
To illustrate this sequence, Scheme 4 shows how the compound of formula (IIa) (II with R5xe2x95x90xe2x80x94Oxe2x80x94C(xe2x95x90NH)xe2x80x94CCl3, P1, P2 and P3=pivaloyl and R6=methyl) is obtained, the synthesis of which is described in the Examples. 
The initial monosaccharide of formula (III) can be obtained from the commercial D-glucosamine hydrochloride of formula (XII), by means of a series of selective hydroxyl protection reactions.
To illustrate this synthetic sequence, Scheme 5 shows how the compound of formula (IIIa) (compound of formula III with P4 and P5=benzylidene, R7=methyl and R8 =acetyl) is obtained, the synthesis of which is described in the Examples.
If R7=benzyl, the anomeric hydroxyl in compound (XIIIa) can be benzylated with benzyl alcohol, in the presence of gaseous hydrogen chloride, and at a temperature between, for example, 50 and 80xc2x0 C. 
Another aspect of the present invention is its utility in human therapy, with application for the prevention or treatment of arthrosis (osteoarthritis), and inflammatory diseases such as inflammatory arthritis, rheumatoid arthritis, psoriatic arthritis, rheumatic fever, palindromic rheumatism, Reiter""s syndrome, lupus eritomatosus and ankylosing spondylitis.
The compounds of the present invention can also be used to control of blood clotting.
This invention also refers to a process to prepare a drug that is effective in the prevention or treatment of arthrosis, of inflammatory diseases and blood clotting, in which said process includes mixing a pharmaceutically acceptable vehicle and a therapeutically effective quantity of a compound of formula (I).
For the therapeutic use of the compounds of the present invention, they are formulated in suitable pharmaceutical compositions, making use of conventional techniques and excipients, as described in Remington""s Pharmaceutical Science Handbook, Mack Pub. Co., N.Y., USA.
The new pharmaceutical compositions of the invention can be administered to patients in suitable doses. The administration of the compositions can be performed in different ways, for example, oral, intravenous, intraperitoneal, intra-articular, subcutaneous, intramuscular, topical, intradermal or intranasal administration. The pharmaceutical compositions of the invention include a therapeutically effective quantity of one of the compounds of the invention. Said quantity depends on a large number of factors such as for example, the physical condition of the patient, age, sex, particular compound, means of administration and other well known factors. It is also understood that the active ingredient can be administered in single or multiple doses, to provide the desired therapeutic effects. If required, other therapeutic agents can be used together with those provided by the present invention.
The compounds of the invention are preferably administered to the patient in a pharmaceutically acceptable vehicle. These vehicles are well known and usually are in solid or liquid form. The pharmaceutical preparations in solid form that can be prepared from this invention include powders, pellets, tablets, dispersible granules, capsules, seals, suppositories and other solid pharmaceutical forms. The preparations in liquid form include solutions, suspensions, emulsions and micro-spheres. Preparations in solid form that can be converted, immediately before use, into preparations in liquid form, for oral, parenteral or intra-articular administration, are also contemplated. Said liquid forms include solutions, suspensions and emulsions.
One advantage of the present invention in relation to glycosaminoglycans glucosaminoglicanes such as hyaluronic acid, lies in the fact that, since the compounds of the present invention are synthetic products, there are no production limitations.
The following examples are not exclusive, and they illustrate the preparation of the compounds of this invention.